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Yeast molecular display is a convenient tool for developing and improving recombinant proteins and assessing their functions. Molecular display using Saccharomyces cerevisiae has been applied for screening valuable biochemical molecules, including antigens and antibodies. In this chapter, we describe the genetic construction of a yeast molecular display system for a nanobody and its evaluation using fluorescence microscopy and flow cytometry.
This study investigated the modulating role of quinoa and chia nanocapsules on the DMBA-induced DNA methylation in rats. Theoretical docking was conducted to assess the binding affinities of selected phytoconstituents from quinoa and chia against the active sites of DNMT1 and TET2. An in vivo study was conducted to determine the effect of nanocapsules on DMBA-induced LINE-1 DNA methylation patterns. Molecular docking suggested that all selected compounds have high binding affinity for DNMT1, with linoleic acid being the top-rate TET2 inhibitor. The in vivo study demonstrated that quinoa nanocapsules can potentially reverse the DMBA-induced LINE-1 methylation pattern in the liver and kidney. Chia nanocapsules did not change the methylation status in the kidney; however, they partially modulated it in the liver. Our findings show that DMBA-induced LINE-1 DNA methylation in the kidney and liver is reversible via quinoa nanocapsules, and is likely driven by their active phytochemicals.
Type 2 diabetes mellitus (T2DM) is a multidimensional metabolic disorder characterized by hyperglycemia, insulin insensitivity, and dysfunction and degeneration of β-cells. Increase in the levels of free fatty acids (FFAs) in blood plasma is a typical symptom of obesity and metabolic syndrome, which influences the mechanisms of insulin resistance and β-cell impairment. Acute increase in FFA levels blocks glucose uptake by insulin-sensitive skeletal muscles while chronic increase in FFA levels results in hepatic insulin resistance which causes gluconeogenic flux and lipotoxicity in pancreatic βcells. FFA-induced insulin resistance involves various molecular pathways and generation of lipid intermediates (diacylglycerol and ceramides), activation of serine/threonine kinases (PKC), induction of oxidative and endoplasmic reticulum stresses, and inflammatory signaling via nuclear factor-κB (NF-κB) and toll-like receptor (TLR) signaling pathways. Recent studies have indicated that the fetuin-A-FFA complex is an important endogenous ligand of TLR4 that causes inflammation and metabolic dysregulation. In addition, certain FFAs, especially ω-6 polyunsaturated fatty acids, can elicit ferroptosis which is a new form of lipid peroxidation-mediated cell death in β-cells, thereby expanding the extent of FFA-mediated lipotoxicity. This review covers recent information on the mechanisms and clinical factors related to the role of FFAs in insulin resistance and T2DM pathogenesis, and the contribution of experimental research towards developing therapeutic strategies in normalizing the levels of FFA and inhibiting downstream lipotoxicity-related pathways.
Cognitive decline is a hallmark of neurodegenerative diseases. The complex pathophysiology of these diseases has limited the efficacy of current therapies. The co-administration of epidermal growth factor (EGF) and growth hormone-releasing peptide 6 (GHRP6) emerges as a promising neuroprotective candidate. The present study evaluated the therapeutic potential of this combination in two preclinical models of cognitive impairment: (i) age-related decline and (ii) impairment induced by intracerebroventricular administration of streptozotocin (STZ). In both experiments, C57BL/6 mice were used and distributed into three experimental groups, each comprising 14-15 animals per group. Cognitive and motor function was assessed through gait pattern, Y-maze and novel object recognition tests. Differential gene expression was analyzed using qPCR. Both models reproduced hallmark features of cognitive decline, including deficits in working and spatial memory and changes in the expression of genes associated with oxidative stress, neuroinflammation and synaptic plasticity. In aged animals, EGF + GHRP6 treatment increased step length (p = 0.04). In the forced alternation Y-maze test, aged-EGF + GHRP6 animals made more visits to the novel arm than to the familiar arm 1 (p = 0.001) or to the familiar arm 2 (p = 0.04). Cognitive benefits were also observed in the STZ-induced model. STZ-EGF + GHRP6 group exhibited an alternation percentage higher than the STZ-vehicle group (p = 0.03). Moreover, EGF + GHRP6 treatment significantly increased the expression of genes associated with antioxidant defense (Hmox1), synaptic plasticity (Creb1), and oligodendrocyte differentiation (Olig1) while concurrently reducing the expression of Nfkb1. These findings highlight the therapeutic potential of EGF + GHRP6 co-administration as a neuroprotective strategy to mitigate neurodegeneration and preserve cognitive function.
Grasslands are among the largest terrestrial biomes and play essential roles in livestock production, carbon sequestration and global food security. The productivity and resilience of these ecosystems are driven by complex molecular interactions between plants and their associated microbiomes. Although recent advances in nucleic acid research and multi-omics approaches have provided new insights into these interactions, the molecular mechanisms underpinning plant-microbiome interactions in these ecosystems remain insufficiently explored. This review synthesizes the latest progress in nucleic-acid and multi-omics approaches to better understand plant-microbiome interactions. It integrates nucleic acid-based technologies with multi-omics frameworks to explain plant-microbiome interactions across molecular, ecological, and management scales. By linking microbial community structure, functional genes, gene expression, metabolite profiles, ecosystem multifunctionality and sustainable grassland management, this review provides a broader framework for translating molecular insights into practical strategies for grassland resilience, productivity, and food security. Advances in amplicon sequencing, shotgun and long-read metagenomics, environmental DNA (eDNA) monitoring, plant and microbiome genome-wide association studies (GWAS) and transcriptomics have provided valuable insights into plant-microbiome interaction. This review highlights how these techniques enable functional and mechanistic understanding by linking microbial diversity with gene expression, nutrient cycling and plant performance. Additionally, long-read sequencing technologies provide genome-resolved analysis, improving the detection of structural and epigenetic variations, which are essential for understanding these interactions. These approaches reveal the role of beneficial microbes in enhancing grassland fertility, ultimately improving grassland productivity. Integrating these findings with metabolomics and phenomics offers a novel approach for predictive modeling in sustainable grassland management. The review concludes by emphasizing the need for standardized protocols, longitudinal field studies and experimental validation through synthetic communities and genome editing to harness plant-microbiome interactions for enhanced productivity and food security.
Polysaccharides and polyphenols frequently coexist in food systems and are consumed simultaneously, yet how these interactions influence polyphenol behavior in the gastrointestinal tract remains insufficiently understood. In this study, the effects of carboxymethyl cellulose (CMC) with different concentrations, molecular weights and degrees of substitution on mucin-tea polyphenol (TP) interactions were systematically investigated. Turbidity and zeta-potential measurements indicated that the addition of CMC reduced aggregation of mucin-TP complexes and increased the negative surface charge. Fluorescence spectroscopy further showed that CMC weakened TP-induced quenching of mucin intrinsic fluorescence, suggesting competitive interactions or conformational interference. Thermodynamic analysis suggested that TP-mucin binding was likely driven primarily by hydrogen bonding and van der Waals forces. Although CMC did not alter the apparent interaction mode, it significantly decreased the binding affinity, particularly under acidic conditions simulating the gastric environment. Moreover, the inhibitory effect became stronger with increasing molecular weight and decreasing degree of substitution of CMC. Molecular docking supported these results, revealing that CMC may compete for mucin binding sites or directly interact with TP molecules. These findings provide molecular-level insights into how food polysaccharides modulate mucin-polyphenol interactions and contribute to a better understanding of the factors affecting interaction behaviors of polyphenols and their gastrointestinal bioavailability. This study may offer useful implications for designing polysaccharide-based delivery or modulation systems for dietary polyphenols in food and health-related applications.
The primary objective of this study is to evaluate whether anesthetic agents modulate the apoptotic activity and migration-inhibitory effects of chemotherapeutic agents, thereby informing the optimization of perioperative management strategies in oncologic interventions. SAOS-2 cells were treated with varying concentrations of thiopental, ketamine, and doxorubicin. Cell viability was assessed via MTT assay to determine IC₅₀ values. Thiopental exhibited greater antiproliferative activity than ketamine and was selected for further study. Its interaction with doxorubicin was analyzed using the Chou-Talalay method, yielding a combination index (CI) of 0.92608 at Fa = 0.5, indicating moderate synergism. The combination treatment also led to reduced IC₅₀ values. Cell migration was evaluated using a scratch wound healing assay. Gene expression changes related to apoptosis and metastasis were analyzed using qRT-PCR. In silico molecular docking simulations were performed to provide mechanistic insights into the observed biological effects. Thiopental and doxorubicin exhibited dose-dependent cytotoxicity, with the combination showing enhanced antiproliferative activity. Co-treatment reduced IC₅₀ values and suppressed SAOS-2 cell migration more effectively than either drug alone. Gene expression analysis revealed increased pro-apoptotic markers and decreased Bcl-2 expression. Metastasis-associated genes were significantly downregulated, despite upregulation of hypoxia-responsive genes. Molecular docking analyses identified interactions of thiopental and doxorubicin with HSP70, HSP90, and CYP450, providing complementary information for the interpretation of the observed cellular responses. The findings suggest that thiopental may influence the cellular response to doxorubicin in osteosarcoma through treatment-associated modulation of apoptosis- and migration-related molecular responses. The observed moderate synergistic interaction and dose-reduction potential warrant further investigation in more clinically relevant experimental models.
Polyethylene (PE) is the most widely produced polyolefin and represents a major contributor to plastic waste, largely due to its chemical stability and resistance to biological degradation. PE biodegradation is typically characterized as a series of oxidative metabolic processes rather than mineralization. An urgent priority is the development of integrated experimental setups that bring together physiological, biochemical, and molecular analyses under controlled cultivation conditions to unlock the metabolic traits of PE biodegradation. This study aims to elucidate the molecular mechanisms of commercial low-molecular-weight PE (LDPE) attack by Rhodococcus opacus R7 through an integrated genome-to-function approach, including growth and extracellular laccase assays, intracellular lipid quantification, gas chromatography coupled with mass spectrometry (GC-MS) analysis of compounds associated with untreated LDPE, and transcriptional profiling of oxidative enzymes. LDPE utilization was evaluated under multiple cultivation strategies (1% single-dose vs. 0.4% fed-batch), inoculum conditions, and polymer types (untreated or UV-pretreated) over 28 days. Fed-batch LDPE (0.4%) supported slightly higher viable cell numbers than fixed-dose LDPE (1%), while prolonged PE pre-adaptation did not enhance growth. Extracellular laccase activity was detected under all conditions, but was more influenced by the physiological status of the inoculum than by PE concentration or pretreatment. Lipid accumulation was early detected in case of 1% PE, while the fed-batch conditions reflected the growth phase and physiological responses. GC-MS analyses revealed that LDPE oxidation products vary compared to abiotic and control samples. Specifically, fixed-dose LDPE favored a progressive change in the pattern-profile of carboxylic acids and medium- to long-chain alkanes, while fed-batch LDPE produced a heterogeneous mixture, including alcohols and ketones, consistent with a continuous and asynchronous transformation of the polymer. The fed-batch condition induced a broader and earlier transcriptional activation of oxidative genes, including seven laccase-like multicopper oxidases (LMCOs), alkane monooxygenase (alkB), benzoate dioxygenase (benA), and cytochrome P450 hydroxylase. Long-chain n-alkanes, particularly tetracosane (C24), strongly induced oxidative enzymes, suggesting their role as metabolic signals during PE degradation. Overall, LDPE attack by R. opacus R7 emerges as a dynamic process shaped by substrate accessibility and cultivation strategy, highlighting its potential as a platform for controlled plastic biodegradation.
In the oleochemical industry, a fat-splitting process is used to generate a methanol-free byproduct, oleochemical industrial waste, that includes crude glycerol. The oleaginous yeast Rhodotorula toruloides is a nonconventional yeast that is well known for its industrial potential as a producer of lipids, carotenoids, and enzymes. Extracellular polysaccharides (EPSs) are of increasing interest due to their many applications. In this study, we found that R. toruloides NBRC 8766 strain produced mannose-rich and high-molecular-weight EPSs with a mannose content (mol%) exceeding 90% and an estimated molecular weight greater than 10,000 k from oleochemical industrial waste crude glycerol or pure glycerol. Components in the industrial waste increased EPS production. The concentration of NH4+ ions in the culture medium was identified as a key factor in discriminating the cells producing the EPS (EPS-producing cells) from EPS-nonproducing cells. Further investigations demonstrated that a sufficient concentration of NH4+ ions (38 mM or more) to acidify the medium to a pH of 2.0 or lower is key to EPS production under these conditions. Therefore, it was hypothesized that the R. toruloides NBRC 8766 strain would produce EPS in response to extracellular acidity. Fluorescent and transmission electron microscopic analyses of the two cell types revealed differences; the EPS-producing cells had immature lipid droplets and marked two-layered cell walls, where the outer layer was darker and looked like it was wearing "fuzz," which was assumed to be the EPSs released from the cells. The EPS-nonproducing cells had clear lipid droplets, one-layered cell walls, and more abundant intracellular polysaccharide granules. In this study, we demonstrated that oleochemical industrial waste is a promising source of crude glycerol for EPS production and that R. toruloides is a promising producer of not only lipids but also EPSs from glycerol. Moreover, the identification of the concentration of NH4+ ions in the culture medium enabled the discrimination of EPS-producing cells from EPS-nonproducing cells. The presence of sufficient NH4+ ions to acidify the medium was demonstrated to be key to EPS production in these conditions. This suggests that the R. toruloides NBRC 8766 strain produces EPS in response to extracellular acidity. The morphological observations of the EPS-producing and EPS-nonproducing cells provide a further basis for understanding the molecular mechanism underlying the production of mannose-rich and high-molecular weight-EPS, as well as clues for artificially increasing the amounts of EPSs and lipids produced.
Conventional molecular diagnostics for imprinting disorders rely on sequential DNA-intensive assays, which are expensive, time-consuming, and often insufficient for detecting mosaicism, resulting in suboptimal clinical management. Here, we present a targeted long-read sequencing strategy that enables the integrated detection of DNA methylation, copy-number variants, and sequence variants in a single assay for Beckwith-Wiedemann spectrum (BWSp), a genomic imprinting disorder caused by genetic or epigenetic alterations affecting imprinting control regions 1 and 2 (IC1/IC2) within the 11p15.5 locus. We evaluated three Oxford Nanopore Technologies (ONT) workflows: adaptive sampling on MinION (AS-MinION), adaptive sampling on PromethION P2 (AS-P2), and whole-genome sequencing on P2 (WGS-P2). Three cases of BWSp patients were analyzed, including two with mosaic paternal uniparental disomy (pUPD) and one with IC2 loss of methylation (LoM). We compared sequencing output, genome-wide and region-of-interest coverage, IC1/IC2 methylation profiles, and variant concordance with Illumina short read sequencing using the Simplex basecalling model. AS-P2 achieved the highest coverage of target regions while maintaining broad and uniform genome-wide coverage, outperforming AS-MinION and WGS-P2. This dual performance enables efficient and scalable simultaneous genetic and epigenetic analysis in a single sequencing run. Using Simplex basecalling, AS-P2 accurately identified all underlying molecular defects in conventionally characterized samples. In conclusion, AS-P2 enablesa cost-effective and sensitive approach for the molecular diagnosis of imprinting disorders, particularly in cases with mosaic or complex genetic and epigenetic architectures.
Proteins are central to nearly all biological processes, mediating enzymatic catalysis, structural scaffolding, and molecular signaling. Deciphering how linear amino acid sequences encode three-dimensional conformations and biological functions has long been a defining challenge in molecular life sciences. Traditional structural biology approaches, such as X-ray crystallography and cryo-electron microscopy, have established the field, yet their high cost, labor intensity, and limited throughput restrict comprehensive coverage of the proteome. The exponential expansion of protein sequence databases, paired with advances in artificial intelligence (AI) and deep learning, has drastically accelerated our ability to predict, annotate, and even design proteins. In this mini review, we trace the evolution of AI-driven methods in protein research, from early residue-contact prediction using coevolutionary information to transformative breakthroughs, the rise of protein language models (PLMs), and the emerging era of generative design and functional modeling. Throughout, we highlight key conceptual advances and their translational implications for biomedical science and biotechnology.
Powdery mildew caused by Golovinomyces cichoracearum is a major constraint in bhendi (Abelmoschus esculentus L.) production, leading to significant yield and quality losses under favourable environmental conditions. This review aims to synthesise current knowledge on the biology, epidemiology and molecular interactions of the bhendi-powdery mildew pathosystem, with emphasis on sustainable management through biological control agents. The pathogen establishes a biotrophic relationship via haustorial development and effector-mediated suppression of host immunity, while host defence involves pattern-triggered immunity and effector-triggered immunity, including reactive oxygen species production and callose deposition. Biological control agents such as Ampelomyces quisqualis, Trichoderma spp. and Bacillus spp. exhibit diverse mechanisms including hyperparasitism, mycoparasitism, antibiosis and induction of systemic resistance. These agents enhance plant defence through increased activity of key enzymes such as peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase, contributing to reduced disease severity. Advances in formulation technologies, including talc-based carriers, alginate encapsulation and oil-assisted delivery systems, have improved the efficacy and field stability of these agents. Integrated approaches combining compatible biological control agents and low-risk chemicals provide enhanced disease suppression compared with single-agent applications. However, variability in field performance, environmental constraints and limited molecular understanding of pathogen diversity remain key challenges. Future strategies integrating omics-based approaches, genome editing and climate-informed disease prediction are essential for improving the consistency and effectiveness of biological control. Overall, biological control integrated within sustainable management frameworks offers a viable alternative to chemical fungicides for managing powdery mildew in bhendi.
Bullfrog (Lithobates catesbeianus) muscle protein remains an underexplored source of bioactive peptides. This study applied an integrated in silico-in vitro-in vivo workflow to identify tyrosinase (TYR)-inhibitory peptides from bullfrog muscle protein hydrolysates. Among five proteases evaluated, acid protease hydrolysis for 3 h produced the highest TYR-inhibitory activity. Following ultrafiltration and Sephadex G-15 gel filtration, the most active fraction (<1 kDa) was analyzed by LC-MS/MS, yielding 71 candidate peptide sequences. The dipeptide FW and tripeptide LAW were prioritized through PeptideRanker scoring, safety prediction, and molecular docking against mushroom TYR (PDB ID: 2Y9X), with predicted docking scores of -8.0 and - 7.9 kcal/mol, respectively. Synthetic FW and LAW exhibited concentration-dependent TYR inhibition, with IC₅₀ values of 0.21 mg/mL (approximately 0.60 mM) and 0.14 mg/mL (approximately 0.36 mM), respectively. Kinetic analysis indicated competitive inhibition by FW and non-competitive inhibition by LAW, while molecular docking suggested distinct interaction patterns broadly consistent with these kinetic profiles. Following simulated gastrointestinal digestion, the corresponding post-digestion samples retained approximately 90% of their initial inhibitory activity, although LAW showed chromatographic evidence of partial degradation or transformation. In zebrafish larvae, neither peptide significantly affected survival or caused apparent developmental abnormalities at 0.0125-0.05 mg/mL, and FW and LAW reduced whole-body melanin content by 30% and 27%, respectively, at 0.05 mg/mL. These findings identify bullfrog muscle protein as a potential source of TYR-inhibitory peptides and support further investigation of FW and LAW for anti-melanogenic applications.
MCL1, an anti-apoptotic BCL-2 family member, is frequently overexpressed in multiple tumour types and correlates with poor prognosis; however, targeting its cytosolic domain to release pro-apoptotic effectors has been limited by cardiotoxicity in clinical trials. In this study, we describe a chemically mediated disruption of the MCL1/BOK transmembrane interaction that induces tumour cell death without affecting viability in 3D cardiomyocyte cultures. Molecular dynamics simulations and LUV-based assays converged to show that the MBoIN179 disrupts transmembrane dimerization, thereby restoring BOK pore formation inhibited by MCL1. In cells, interference with the MCL1/BOK transmembrane interaction promoted relocalization of BOK from the endoplasmic reticulum to mitochondria, where it engaged a BOK-dependent cell death program in both 2D and 3D breast cancer models, leading to reduced tumour growth and metastasis in vivo. Analysis of patient tumour microarrays further showed that BOK is overexpressed in aggressive breast cancer subtypes and correlates with poor prognosis. Together, these findings identify the MCL1/BOK transmembrane interaction as a tumour-selective vulnerability that can be engaged to promote antitumour responses with cardiac safety, highlighting transmembrane interactions as viable molecular targets.
Milk protein synthesis in goats is governed by multilevel regulatory mechanisms coordinating genetic, hormonal, nutritional, and cellular signals. Despite growing interest in goat milk proteins due to their distinctive nutritional properties, digestibility, and reduced allergenicity, their regulatory basis remains less characterised than other dairy species. This review integrates current knowledge on goat milk protein composition, genetic polymorphism, and molecular regulation, summarising structural and functional characteristics of caseins, whey proteins, and minor bioactive proteins. It examines how polymorphisms and haplotypes contribute to breed-specific variation in milk yield, composition, and physicochemical properties, highlighting JAK-STAT and mTOR signalling pathways' central roles in integrating hormonal and nutritional signals that control transcription and translation. Evidence from GWAS, QTL analyses, transcriptomics, proteomics, and epigenetic studies outline the genomic architecture and stage-specific regulation of milk protein synthesis, while highlighting knowledge gaps and research opportunities. By integrating genomic, transcriptomic, proteomic, and epigenetic evidence, this review highlights candidate regulatory variants and molecular pathways underlying milk protein synthesis, providing a framework for functional validation, precision breeding, and the development of high-value goat dairy products.
Poecilobdella manillensis Lesson is a well-recognized medicinal leech in traditional Chinese medicine and Guangxi Zhuang ethnic medicine. It has long been used to activate blood circulation and remove blood stasis for the treatment of ischemic stroke. Modern pharmacological research has verified its potent anticoagulant and anti-inflammatory activities. Current studies mainly focus on its polypeptide components that exert antithrombotic effects to improve cerebral ischemia, while the neuroprotective potential and related mechanisms of its small-molecule constituents remain largely unclear. This study aimed to investigate the therapeutic effects of the ethyl acetate extract (EA) of P. manillensis on cerebral ischemia-reperfusion injury and to clarify its underlying molecular mechanism. The chemical constituents of EA were identified by UPLC-Q-TOF-MS/MS. Network pharmacology and molecular docking were used to predict and verify core targets and pathways. Neuroprotective and anti-inflammatory effects of EA were evaluated in a rat MCAO/R model, OGD/R-injured SH-SY5Y cells, and LPS-stimulated BV2 cells, using histological staining, Western blot, immunohistochemistry, and RT-qPCR. Seven small-molecule components were identified in EA, and 314 overlapping targets related to ischemic stroke were screened. Network analysis showed that TLR4 was the core target, and the main enriched pathways included NF-κB, Toll-like receptor, apoptosis and TNF signaling pathways. Consistent with the predicted results, EA significantly reduced cerebral infarct volume and improved neurological deficits in MCAO/R rats, and inhibited neuronal apoptosis and microglial inflammation in vivo. In vitro, EA notably improved the survival of OGD/R-injured neurons and suppressed LPS-induced inflammatory responses in BV2 cells. Meanwhile, EA markedly downregulated the expression of TLR4/NF-κB and NLRP3 inflammasome-related molecules. The present study demonstrated that EA protects against cerebral ischemia-reperfusion injury by inhibiting neuronal apoptosis and TLR4/NF-κB-mediated neuroinflammation. These findings provide a scientific basis for the traditional clinical application of P. manillensis and suggest that EA could serve as a potential therapeutic candidate for ischemic stroke.
Human norovirus (HuNoV) and hepatitis A virus (HAV) are highly prevalent and contagious foodborne pathogens that pose a significant threat to global public health. Current molecular detection methods such as RT-qPCR and RT-ddPCR are highly sensitive and specific but time-consuming, require specialized equipment, and are unsuitable for on-site detection. We developed a multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay coupled with CRISPR-Cas12a and lateral flow detection for rapid, simultaneous identification of HuNoV GI, GII, and HAV. Through rigorous in silico design and experimental validation, we optimized primer pools and crRNAs to ensure broad genotype coverage and high specificity. Using 2 µL of input per target per 50 µL reaction, the assay achieved limits of detection of 10¹ copies/µL (2 × 10¹ copies/reaction) for HAV, 10³ copies/µL (2 × 10³ copies/reaction) for GI HuNoV, and 10² copies/µL (2 × 10² copies/reaction) for GII HuNoV, with a total assay time of 50 min from purified RNA to final readout. No cross-reactivity occurred with other common foodborne viruses. Validation using total RNA extracted from shellfish digestive glands artificially spiked with RNA standards provided preliminary evidence supporting the feasibility of the method under laboratory conditions. This portable system shows strong potential as a rapid multiplex molecular detection platform.
This study introduces a novel cold-active alkaline protease-producing Bacillus mobilis TK31 strain, first isolated from Türkiye and identified through biochemical and molecular analyses. Combined mutagenesis using UV irradiation and ethyl methanesulfonate (EMS) enhanced protease production of the strain under optimized conditions. Proteases from wild-type and mutant strains were partially purified by acetone precipitation and subjected to preliminary comparative characterization. Both enzymes exhibited optimal activity at a pH of 9.0 and a temperature of 15 °C. The mutant 18C-179 enzyme showed a 3.75-fold activity increase over the wild-type post-optimization. The enzymes' pH and temperature stability, chemical resistance, and substrate specificity were evaluated, with the mutant displaying superior stability at extreme pH values, higher temperatures, and enhanced resistance to specific chemicals. Casein hydrolysis products were identified chromatographically. The wild-type and mutant enzymes demonstrated promising potential in detergent, photography, and pharmaceutical applications. This work presents a cold-active alkaline protease and highlights its potential for further industrial biotechnology studies through strain improvement.
Aging entails complex physiological changes, yet large-scale evidence among older Japanese individuals, especially those with comorbidities, remains limited. We analyzed serum and plasma samples from approximately 3800 Japanese aged 40 years and older to identify age-associated proteins and lipids, focusing on reproducibility and robustness. Chemokines CXCL9 and CCL11 and phosphatidylcholines PC 31:0 and PC 32:0 were positively associated with age across five cohorts, whereas lysophosphatidylcholines LPC-LA and LPC-AA showed negative associations. These molecular relationships were consistently reproduced across serum and plasma matrices and replicated in independent cohorts. Cross-platform consistency was confirmed between Olink Target 96 (relative NPX) and Target 48 (absolute quantification), with direct validation in Cohort 2. To our knowledge, this is the largest study to demonstrate reproducibility of age-associated molecular biomarkers in a comorbidity-enriched Japanese population. The principal contribution is technical─defining a set of robust, cross-platform, cross-matrix biomarkers of aging in older adults. Unlike previous Western studies which focused on younger or healthier populations, this work establishes reproducibility and generalizability in real-world aging. These validated biomarkers provide a valuable reference for clinical and translational research, including risk stratification and biological age assessment in comorbidity-enriched settings.
Chromosomal triplications are rare structural variations often associated with complex phenotypes. We report the molecular characterization of a novel intrachromosomal triplication at 18q12.1q21.2 identified in a fetus with ultrasound abnormalities. Conventional karyotyping and array-CGH revealed a partial tetrasomy and a 26 Mb region of loss of homozygosity (LOH), extending from the triplication to the telomere. Long-read sequencing (LRS) identified breakpoint junctions revealing a direct-inverted-direct triplication structure. Breakpoint analysis suggested that this rearrangement arose through a U-type exchange between sister chromatids, likely mediated by microhomology-based mechanisms. This process likely generated a transient dicentric chromosome that subsequently broke during mitosis. The resulting duplicated chromosome may have been stabilized by telomere capture, consistent with the triplicated 18q12.1q21.2 region followed by the 18q21.2q23 LOH. Nine genes within the triplicated region, including SMAD2 and SMAD4, showed high predicted sensitivity to increased dosage, possibly contributing to the clinical phenotype. This study highlights the utility of LRS in defining complex chromosomal rearrangements and emphasizes the importance of molecular breakpoint analysis for understanding pathogenic mechanisms and improving genetic prenatal diagnosis.